Tumwebaze et al. Parasites Vectors (2019) 12:565 https://doi.org/10.1186/s13071-019-3811-2
RESEARCH
Molecular identification of Bulinus spp. intermediate host snails of Schistosoma spp. in crater lakes of western Uganda with implications for the transmission of the Schistosoma haematobium group parasitesImmaculate Tumwebaze1*, Catharina Clewing1, Marie Claire Dusabe2, Julius Tumusiime3, Grace Kagoro‑Rugunda3, Cyril Hammoud4,5 and Christian Albrecht1,3
Abstract
Background: Human schistosomiasis is the second most important tropical disease and occurs in two forms in Africa (intestinal and urogenital) caused by the digenetic trematodes Schistosoma mansoni and Schistosoma haematobium, respectively. A proposed recent shift of schistosomiasis above a previously established altitudinal threshold of 1400 m above sea level in western Ugandan crater lakes has triggered more research interest there.
Methods: Based on extensive field sampling in western Uganda and beyond and employing an approach using sequences of the mitochondrial barcoding gene cytochrome c oxidase subunit 1 (cox1) this study aims were: (i) iden‑tification and establishment of the phylogenetic affinities of Bulinus species as potential hosts for Schistosoma spp.; (ii) determining diversity, frequency and distribution patterns of Bulinus spp.; and (iii) establishing genetic variability and phylogeographical patterns using Bayesian inference and parsimony network analyses.
Results: Out of the 58 crater lakes surveyed, three species of Bulinus snails were found in 34 crater lakes. Bulinus tropicus was dominating, Bulinus forskalii was found in two lakes and Bulinus truncatus in one. The latter two species are unconfirmed potential hosts for S. haematobium in this region. However, Bulinus tropicus is an important species for schistosomiasis transmission in ruminants. Bulinus tropicus comprised 31 haplotypes while both B. forskalii and B. truncatus exhibited only a single haplotype in the crater lakes. All species clustered with most of the haplotypes from surrounding lake systems forming source regions for the colonization of the crater lakes.
Conclusions: This first detailed malacological study of the crater lakes systems in western Uganda revealed pres‑ence of Bulinus species that are either not known or not regionally known to be hosts for S. haematobium, the causing agent of human urogenital schistosomiasis. Though this disease risk is almost negligible, the observed dominance of B. tropicus in the crater lakes shows that there is a likelihood of a high risk of infections with Schistosoma bovis. Thus, extra attention should be accorded to safeguard wild and domestic ruminants in this region as the population ben‑efits from these animals.
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Open Access
Parasites & Vectors
*Correspondence: [email protected]; [email protected]‑giessen.de1 Department of Animal Ecology and Systematics, Justus Liebig University Giessen, Giessen, GermanyFull list of author information is available at the end of the article
Page 2 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
BackgroundSchistosomiasis is an important tropical disease espe-cially in sub-Saharan Africa, with more than 90% of the disease burden [1] and the second most important pub-lic health disease after malaria [1, 2]. Schistosomiasis is a parasitic disease transmitted by planorbid gastropods. Human schistosomiasis in Africa occurs in two forms (intestinal and urogenital), caused by the digenetic trem-atodes Schistosoma mansoni and Schistosoma haemato-bium, respectively. Urogenital schistosomiasis accounts officially for two-thirds of all cases [3], a figure that might be too optimistic as the real prevalence of the disease is potentially underestimated by a factor of three [4]. The already important direct impact of urogenital schisto-somiasis is worsened by its established role in cancer epidemics and AIDS epidemics in Africa (reviewed in [5]), besides the long recognized roles in other patholo-gies and diseases such as haematuria and female genital schistosomiasis (reviewed in [6]). Interestingly, S. haema-tobium is the least studied of the major human schisto-somes [7, 8].
Unlike in many other regions of sub-Saharan Africa, in Uganda, intestinal rather than urogenital schistosomiasis
is considered a major public health problem [9]. Uro-genital schistosomiasis, though present has long been assumed to be restricted to a few areas of eastern and northern Uganda [10]. Schistosomiasis studies in Uganda have so far been focused on regional intestinal schisto-somiasis [11–14] around the great lake systems of Lake Victoria and Lake Albert. A negligible amount of studies have been conducted on urogenital schistosomiasis and S. haematobium and their planorbid host snails belong-ing to the genus Bulinus. Today there is no official decla-ration about any region of Uganda to be completely free of urogenital schistosomiasis. Thus, the status of urogeni-tal schistosomiasis remains an enigma in Uganda.
A recent study [15] has indicated that intestinal schis-tosomiasis actually occurs above an earlier designated threshold of 1400 m above sea level (a.s.l.), specifically in crater lakes in western Uganda. For instance, a high rate of intestinal schistosomiasis in travelers after a brief exposure to the high-altitude crater Lake Nyinam-buga was reported in 2012 [16]. The Albertine Rift val-ley region of western Uganda is dominated by mainly two types of freshwater bodies; the three great lakes Albert, Edward, George and about 90 small crater lakes of
Keywords: Bulinus forskalii, Bulinus tropicus, Bulinus truncatus, Schistosoma haematobium, Schistosoma bovis, Neglected tropical disease, Schistosomiasis surveillance
1000 km
Equator
Africa
5 km
L. Edward
Kazinga channel
L. George
2 km
Kiba
le fo
rest
Rwen
zori
mou
ntain
rang
es
N
B. truncatusB. tropicus
B. forskaliiB. africanus groupNo Bulinus snails found
100 km
L. K
ivu
L. Victoria
L. Turkana
L. Tanganyika
L. Kyoga
Indian
Oce
an
L. Albert
Uganda Kenya
Tanzania
BurundiRwanda
a
b
c d
Maramagambo forest
2 km
Rwen
zori
moun
tain r
ange
s
Fort Portal town
d
Fig. 1 Sampling sites in the three crater lakes fields in Uganda and at the supra‑regional scale. a Ndali‑Kasenda crater lakes. b Bunyaruguru crater lakes. c Populations from East Africa. d Fort Portal crater lakes. Snail symbols indicate localities of Bulinus populations studied, whereas a star indicates when sampling did not yield a Bulinus population in the respective crater lake. Note that data for some populations used were retrieved from GenBank (see Table 1)
Page 3 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
varying sizes scattered throughout the region. The crater lakes have originally been divided into four geographical fields of Fort Portal, Ndali-Kasenda, Katwe-Kikorongo and Bunyaruguru [17] (see Fig. 1). They straddle the equator between and are spread along the regional rift valley gradient from 914 m to 1566 m elevation [18], with varying limnological characteristics [19] and climatic gradient. The region is one of the most densely populated rural areas in sub-Saharan Africa [20] and is also a tourist destination, attracting local and international travelers.
The potential of urogenital schistosomiasis in Uganda has been neglected, despite the fact that the disease is common in regions nearby such as the Democratic Republic of the Congo (DRC) [21], Tanzania [22] and South Sudan [23]. It has recently been shown that target-ing the schistosome intermediate hosts is the most effec-tive of all elimination strategies combating the burden of schistosomiasis [24]. A first step in targeting regional transmission foci is the correct identification of the intermediate hosts [25]. This is particularly true for the Bulinus spp./Schistosoma haematobium system, since Bulinus is a very diverse freshwater gastropod genus of currently recognized 37 species belonging to four spe-cies complexes that are morphologically variable [26, 27]. Although there are still some taxonomic issues involved, it has repeatedly been shown that these species com-plexes can be identified using molecular genetic tools [28–31]. Three of the species complexes are known to occur in regions along the Albertine Rift, namely the B. truncatus/B. tropicus complex, the B. forskalii group and the B. africanus group [32]. These regions are potentially species source pools for the crater lakes. Given that these groups include potential hosts for S. haematobium, it is important to survey the crater lakes region for snails transmitting human urogenital schistosomiasis. There-fore, enhanced simultaneous mapping and monitoring of strains of urogenital schistosomiasis and their intermedi-ate hosts populations are both necessary to control this disease in areas where it has not been known to occur before. Very little information, however, exists on the mollusc fauna of the crater lakes [33]. This necessitates an assessment of the status of potential host snail species and thus urogenital schistosomiasis in the region.
Based on extensive field sampling in the crater field region and beyond and employing an approach using sequences of the barcoding gene of mitochondrial cytochrome c oxidase subunit 1 (cox1) this study aims are: (i) identification and establishment of the phyloge-netic affinities of Bulinus species as potential hosts for Schistosoma spp.; (ii) determining diversity, frequency and distribution patterns of Bulinus spp.; and (iii) establishing genetic variability and phylogeographical patterns.
MethodsStudy areaThis study was conducted in lakes of the three main crater fields in Uganda, between Fort Portal region in the north, Ndali-Kasenda in the middle and Bunyaru-guru in the south (Fig. 1). The region is bordered by the vast Rwenzori Mountains in the north-west, the southern shores of Lake Albert in the north and the region of the Queen-Elizabeth National Park (Lake Edward-George) in the south. Most of the crater lakes were formed by faulting and volcanic eruption some 8000 to 10,000 years ago [34]. Bunyaruguru lakes lie on the southern side of the Edward-George system while the rest are located on its northern side. The climate, hydrology, water chemistry and landscape settings are highly heterogeneous between crater fields. Lakes in Fort Portal crater field lie at higher altitude (above 1500 m a.s.l), than those in the Ndali-Kasenda crater field and Bunyaruguru. Lake Kyaninga (Fig. 2a) is one of the deepest (220 m) known crater lake in western Uganda [35], although the crater lakes are generally shallow. Some of the lakes are embedded in a still rather natural setting whereas the surroundings of many of the lakes studied are highly disturbed by anthropogenic activities. The lakes are commonly exploited as a water source for humans and livestock consumption (Fig. 2). Furthermore, these lakes are also a main source of food for the local communities using them for fishing. Some of the fish species are introduced. For example, Tilapia zillii, Orechromis leucosticus and Poecilia reticulata were introduced in Lake Nkuruba [36].
SamplingOut of 58 crater lakes sampled, 19 were from Bun-yaruguru, 33 from Ndali-Kasenda and the 6 remaining from Fort Portal crater field (Table 1). We purposively selected these lakes to cover a range from low altitude (1033 m a.s.l) to high altitude (1569 m a.s.l). The selec-tion of a lake and/or sampling site was based on rep-resentation of lake field size, lake size class, utilization and lake type as well as accessibility. Lakes of Katwe-Kikorongo field are known to be mainly saline [19] and were therefore not included in this study.
Since the access to the crater lakes is often made dif-ficult by their steep escarpments, we collected snails from one to two localities per crater lake. We used scoop netting and/or dredging sampling techniques to capture snails along the edges of the access point of the lake. A maximum of 40 min sampling time per lake was used. Sampling also involved visual inspection of shoreline vegetation and hand-picking of snails. Sam-ples were derived from depths down to a maximum
Page 4 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
of 1.5 m, covering all major habitat types present. The collected snails were fixed in 80% ethanol and stored at −20 °C for subsequent genetic analyses.
DNA isolation, amplification and sequencingPrior to DNA isolation, we photographed all specimens with a digital microscope system (KEYENCE VHX-2000; Keyence Deutschland GmbH, Neu-Isenburg, Germany). Genomic DNA was isolated using the CTAB method of DNA extraction [37]. In a few cases, DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen, Mississauga,
ON, Canada) following the provided instructions. A frag-ment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) with a target length of 655 bp was amplified using the Folmer region primers LCO1490 [38] and COR722B [39]. PCR reactions were run according to Albrecht et al. [37]. Sanger DNA sequencing was performed on an ABI 3730xl DNA analyzer using the BigDye Terminator Kit (Life Technologies, LGC Genomics GmbH, Berlin, Ger-many). Vouchers (shells and DNA) are deposited in the University of Giessen Systematics and Biodiversity col-lection (UGSB, [40]).
Fig. 2 Impressions from selected crater lakes in western Uganda. a Lake Kyaninga (Fort Portal). b Lake Nyinambuga (Ndali‑Kasenda). c Lake Ekikoto (Fort Portal). d Lake Ntambi (Ndali‑Kasenda). e Lake Nyamugosani (Ndali‑Kasenda). f Lake Kayihara (Fort Portal). g Lake Kako (Bunyaruguru) (Photo credit: C. Albrecht (a, c, f); D. Engelhardt (b); C. Dusabe (d, e); I. Tumwebaze (g))
Page 5 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
Sum
mar
y on
loca
litie
s an
d ha
plot
ypes
foun
d in
the
crat
er la
kes
and
othe
r reg
ions
stu
died
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
Buny
arug
uru
Lake
Bug
wag
yi, U
gand
aBG
J−
0.19
680°
N30
.186
63°E
1070
B. tr
opic
us1
BGJ1
.1U
GSB
213
0225
644
MN
5515
18
B. tr
opic
usBG
J1.2
UG
SB 2
1303
2564
5M
N55
1519
Lake
Che
mab , U
gand
a−
0.25
229°
N30
.114
44°E
1268
––
––
Lake
Kab
arog
yib , U
gand
a−
0.22
692°
N30
.213
75°E
1352
––
––
Lake
Kak
ob , Uga
nda
−0.
3048
4°N
30.0
9728
°E14
05–
––
–
Lake
Kam
unzu
kub , U
gand
a−
0.26
422°
N30
.154
10°E
1272
––
––
Lake
Kam
wer
ub , Uga
nda
−0.
2602
3°N
30.1
2223
°E12
70–
––
–
Lake
Kar
iyab , U
gand
a−
0.23
010°
N30
.166
24°E
1256
––
––
Lake
Kas
iriya
b , Uga
nda
−0.
2567
3°N
30.1
3180
°E12
65–
––
–
Lake
Kat
inda
b , Uga
nda
−0.
2223
7°N
30.1
0978
°E10
33–
––
–
Lake
Kig
ezi,
Uga
nda
KGZ
−0.
2858
9°N
30.1
1084
°E13
23B.
trop
icus
1KG
Z1.1
UG
SB 2
1308
2564
8M
N55
1520
B. tr
opic
usKG
Z1.2
UG
SB 2
1309
2564
9M
N55
1521
Lake
Kya
mw
iga,
Uga
nda
KMG
−0.
1919
6°N
30.1
5009
°E10
35B.
trop
icus
1KM
G1.
1U
GSB
212
9625
640
MN
5515
16
B. tr
opic
usKM
G1.
2U
GSB
212
9725
641
MN
5515
17
Lake
Kya
sand
uka,
Uga
nda
−0.
2874
5°N
30.0
4731
°E10
16B.
trun
catu
sU
GSB
236
2827
178
MN
5515
75
B. tr
unca
tus
UG
SB 2
3629
2717
9M
N55
1576
Lake
Maf
uro,
Uga
nda
MFR
−0.
2674
9°N
30.1
0385
°E12
79B.
trop
icus
3M
FR1.
1U
GSB
212
8725
634
MN
5515
10
B. tr
opic
usM
FR1.
2U
GSB
212
8825
635
MN
5515
11
B. tr
opic
usM
FR2.
1H
Q12
1571
[32]
Lake
Mug
ogob , U
gand
a−
0.28
262°
N30
.125
91°E
1323
–
Lake
Mur
ambi
, Uga
nda
MRM
−0.
2251
2°N
30.1
0789
°E10
79B.
trop
icus
MRM
1.1
UG
SB 2
1290
2563
6M
N55
1512
B. tr
opic
usM
RM1.
2U
GSB
212
9125
637
MN
5515
13
Lake
Nya
mus
ingi
reb , U
gand
a−
0.28
455°
N30
.039
94°E
985
––
––
Lake
Nku
gute
b , Uga
nda
−0.
3206
8°N
30.1
0341
°E14
04–
––
–
Lake
Nyu
ngu,
Uga
nda
NG
U−
0.25
500°
N30
.095
24°E
1196
B. tr
opic
us2
NG
U1.
1U
GSB
212
9325
638
MN
5515
13
B. tr
opic
usN
GU
1.2
UG
SB 2
1294
2563
9M
N55
1515
B. tr
opic
usN
GU
2.1
HQ
1215
71 [3
2]
Lake
Rw
ijong
oc , Uga
nda
RWG
−
0.27
122°
N30
.089
09°E
1262
B. tr
opic
us2
RWG
2.2
HQ
1215
70 [3
2]
B. tr
opic
usRW
G2.
3H
Q12
1571
[32]
Nda
li Ka
send
aLa
ke K
anya
bute
rere
b , Uga
nda
0.41
485°
N30
.288
23°E
1201
––
––
Lake
Kan
yam
ukal
i, U
gand
aKM
L0.
4069
9°N
30.2
3669
°E11
67B.
trop
icus
2KM
L1.1
UG
SB 2
1434
2583
9M
N55
1526
B. tr
opic
usKM
L1.2
UG
SB 2
1435
2584
0M
N55
1527
B. tr
opic
usKM
L1.3
UG
SB 1
6767
2252
3M
N55
1503
Page 6 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
B. tr
opic
usKM
L1.4
UG
SB 1
6768
2252
4M
N55
1504
B. tr
opic
usKM
L1.5
UG
SB 1
6853
2257
1M
N55
1508
Lake
Kan
yang
we,
Uga
nda
KAG
0.45
041°
N30
.275
86°E
1280
B. tr
opic
us3
KAG
1.1
UG
SB 2
2111
2633
5M
N55
1554
B. tr
opic
usKA
G1.
2U
GSB
221
1226
336
MN
5515
55
B. tr
opic
usKA
G2.
1H
Q12
1576
[32]
Lake
Kas
enda
, Uga
nda
KSD
0.43
285°
N30
.291
79°E
1248
B. tr
opic
us5
KSD
1.1
UG
SB 2
1431
2583
7M
N55
1524
B. tr
opic
usKS
D1.
2U
GSB
214
3225
838
MN
5515
25
B. tr
opic
usKS
D2.
1H
Q12
1577
[32]
B. tr
opic
usKS
D2.
2H
Q12
1578
[32]
B. tr
opic
usKS
D2.
3H
Q12
1579
[32]
Lake
Kib
ungo
, Uga
nda
KIG
0.39
237°
N30
.233
38°E
1140
B. fo
rska
lii1
KIG
1.1
UG
SB 2
2375
2662
0M
N55
1573
Lake
Kifu
ruka
b , Uga
nda
0.48
912°
N30
.288
45°E
1407
––
––
Lake
Kite
re, U
gand
aKT
R0.
3973
1°N
30.2
7346
°E11
44B.
trop
icus
2KT
R1.1
UG
SB 2
1570
2595
6M
N55
1541
B. tr
opic
usKT
R1.2
UG
SB 2
1571
2595
7M
N55
1542
Lake
Kya
nga,
Uga
nda
KYG
0.40
022°
N30
.232
21°E
1167
B. tr
opic
us2
KYG
1.1
UG
SB 2
1455
2585
3M
N55
1532
B. tr
opic
usKY
G1.
2U
GSB
214
5625
854
MN
5515
33
Lake
Lug
embe
, Uga
nda
LGB
0.44
722°
N30
.281
23°E
1298
B. tr
opic
us1
LGB1
.1U
GSB
221
2326
343
MN
5515
60
B. tr
opic
usLG
B1.2
UG
SB 2
2124
2634
4M
N55
1561
Lake
Lya
nton
deb , U
gand
a0.
4866
2°N
30.2
8024
°E14
04–
––
–
Lake
Mira
mbi
, Uga
nda
MRB
0.38
902°
N30
.229
06°E
1144
B. fo
rska
lii1
MRB
1.1
UG
SB 2
2120
2634
1M
N55
1559
B. fo
rska
liiM
RB1.
2U
GSB
223
7226
618
MN
5515
74
Lake
Mub
irob , U
gand
a0.
4414
4°N
30.2
5545
°E12
12–
––
–
Lake
Mul
igam
ire, U
gand
aM
RR0.
4230
2°N
30.2
8884
°E12
08B.
trop
icus
1M
RR1.
1U
GSB
215
6125
950
MN
5515
38
B. tr
opic
usM
RR1.
2U
GSB
215
6225
951
MN
5515
39
Lake
Mw
amba
, Uga
nda
MBA
0.45
746°
N30
.273
03°E
1307
B. tr
opic
us4
MBA
1.1
UG
SB 2
2093
2632
3M
N55
1547
B. tr
opic
usM
BA1.
2U
GSB
223
6426
610
MN
5515
71
B. tr
opic
usM
BA1.
3U
GSB
223
6526
611
MN
5515
70
B. tr
opic
usM
BA2.
1H
Q12
1575
[32]
Lake
Mw
enge
nyi,
Uga
nda
MG
Y0.
4875
7°N
30.2
5972
°E14
10B.
trop
icus
2M
GY1
.1U
GSB
221
1726
339
MN
5515
57
B. tr
opic
usM
GY1
.2U
GSB
221
1826
340
MN
5515
58
Lake
Ndi
chob , U
gand
a0.
4452
5°N
30.2
6904
°E12
72–
––
–
Lake
Nja
raya
bana
, Uga
nda
NJN
0.42
805°
N30
.247
47°E
1204
B. tr
opic
us2
NJN
1.1
UG
SB 2
2366
2661
2M
N55
1569
B. tr
opic
usN
JN1.
2U
GSB
223
6726
613
MN
5515
68
Page 7 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
Lake
Nku
ruba
, Uga
nda
NRB
0.51
720°
N30
.303
24°E
1517
B. tr
opic
us6
NRB
1.1
UG
SB 2
1549
2594
2M
N55
1534
B. tr
opic
usN
RB1.
2U
GSB
215
5025
943
MN
5515
35
B. tr
opic
usN
RB1.
3U
GSB
167
6122
517
MN
5515
01
B. tr
opic
usN
RB1.
4U
GSB
167
6222
518
MN
5515
02
B. tr
opic
usN
RB2.
1H
Q12
1568
[32]
B. tr
opic
usN
RB2.
2H
Q12
1567
[32]
B. tr
opic
usN
RB2.
3H
Q12
1566
[32]
B. tr
opic
usN
RB2.
4H
Q12
1569
[32]
Lake
Nta
mbi
b , Uga
nda
0.40
729°
N30
.229
46°E
1155
––
––
Lake
Nta
nda/
Kata
nda,
Uga
nda
KTD
0.47
775°
N30
.261
65°E
1341
B. tr
opic
us1
KTD
1.1
UG
SB 2
2108
2633
3M
N55
1552
B. tr
opic
usKT
D1.
2U
GSB
221
0926
334
MN
5515
53
Lake
Nya
bike
re, U
gand
aN
KR0.
5010
1°N
30.3
2792
°E13
89B.
trop
icus
3N
KR1.
1U
GSB
214
4025
843
MN
5515
28
B. tr
opic
usN
KR1.
2U
GSB
214
4125
844
MN
5515
29
B. tr
opic
usN
KR2.
1H
Q12
1572
[32]
B. tr
opic
usN
KR2.
2H
Q12
1574
[32]
Lake
Nya
hira
, Uga
nda
NH
R0.
4991
4°N
30.2
8737
°E14
58B.
trop
icus
1N
HR1
.1U
GSB
215
5225
944
MN
5515
36
B. tr
opic
usN
HR1
.2U
GSB
215
5325
945
MN
5515
37
Lake
Nya
miri
ma,
Uga
nda
NM
M0.
5200
1°N
30.3
2035
°E14
97B.
trop
icus
2N
MM
1.1
UG
SB 2
1720
2608
0M
N55
1543
B. tr
opic
usN
MM
1.2
UG
SB 2
1721
2608
1M
N55
1544
B. tr
opic
usN
MM
2.1
HQ
1215
68 [3
2]
Lake
Nya
mug
osan
i, U
gand
aN
GS
0.42
498°
N30
.231
39°E
1237
B. tr
opic
us2
NG
S1.1
UG
SB 2
1428
2583
5M
N55
1522
B. tr
opic
usN
GS1
.2U
GSB
214
2925
836
MN
5515
23
B. tr
opic
usN
GS1
.3U
GSB
220
9926
327
MN
5515
50
Lake
Nya
mug
orob , U
gand
a0.
4480
9°N
30.2
4233
°E12
72–
––
–
Lake
Nya
mut
eza,
Uga
nda
NTZ
0.43
509°
N30
.228
66°E
1261
B. tr
opic
us2
NTZ
1.1
UG
SB 2
1734
2608
9M
N55
1545
B. tr
opic
usN
TZ1.
2U
GSB
217
3526
090
MN
5515
46
Lake
Nya
nsw
iga,
Uga
nda
NSG
0.50
733°
N30
.288
25°E
1479
B. tr
opic
us1
NSG
1.1
UG
SB 2
1567
2595
4M
N55
1540
B. tr
opic
usN
SG1.
2U
GSB
223
5926
606
MN
5515
72
Lake
Nyi
nabu
lita,
Uga
nda
NBT
0.50
760°
N30
.325
33°E
1424
B. tr
opic
us2
NBT
1.1
UG
SB 2
1446
2584
7M
N55
1530
B. tr
opic
usN
BT1.
2U
GSB
214
4725
848
MN
5515
31
Lake
Nyi
nam
buga
, Uga
nda
NN
G0.
4810
9°N
30.2
8773
°E13
72B.
trop
icus
3N
NG
1.1
UG
SB 2
2106
2633
2M
N55
1551
B. tr
opic
usN
NG
1.2
UG
SB 2
2368
2661
4M
N55
1567
B. tr
opic
usN
NG
1.3
UG
SB 2
2369
2661
5M
N55
1566
Page 8 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
B. tr
opic
usN
NG
1.4
UG
SB 1
7784
2316
0M
N55
1500
B. tr
opic
usN
NG
2.1
HQ
1215
73 [3
2]
Lake
Ruk
wan
zi, U
gand
aRK
Z0.
4748
1°N
30.2
7925
°E13
45B.
trop
icus
1RK
Z1.1
UG
SB 2
2115
2633
8M
N55
1556
B. tr
opic
usRK
Z1.2
UG
SB 2
2370
2661
6M
N55
1565
B. tr
opic
usRK
Z1.3
UG
SB 2
2371
2661
7M
N55
1564
Lake
Rw
anda
kara
, Uga
nda
RKR
0.41
567°
N30
.270
54°E
1171
B. tr
opic
us2
RKR1
.1U
GSB
221
2626
345
MN
5515
62
B. tr
opic
usRK
R1.2
UG
SB 2
2127
2634
6M
N55
1563
Lake
Rw
enju
ba, U
gand
aRJ
B0.
4392
2°N
30.2
6577
°E12
57B.
trop
icus
2RJ
B1.1
UG
SB 2
2096
2632
5M
N55
1548
B. tr
opic
usRJ
B1.2
UG
SB 2
2097
2632
6M
N55
1549
Lake
Wan
kenz
i, U
gand
aW
KZ0.
4183
4°N
30.2
6326
°E11
58B.
trop
icus
3W
KZ1.
1U
GSB
167
7022
526
MN
5515
05
B. tr
opic
usW
KZ1.
2U
GSB
168
3922
557
MN
5515
07
B. tr
opic
usW
KZ1.
3U
GSB
168
5422
572
MN
5515
09
Fort
Por
tal
Lake
Bal
amab , U
gand
a0.
6795
9°N
30.2
3345
°E15
66–
––
Lake
Eki
koot
ob , Uga
nda
0.70
142°
N30
.313
42°E
1536
––
–
Lake
Kay
ihar
ab , Uga
nda
0.70
256°
N30
.315
90°E
1549
––
–
Lake
Kya
ning
a, U
gand
a0.
7007
0°N
30.2
9919
°E15
53–
––
Lake
Saa
kab , U
gand
a0.
6870
0°N
30.2
3954
°E15
69–
––
Lake
Wab
iker
eb , Uga
nda
0.68
826°
N30
.235
18°E
1550
––
–
Oth
ers
(non
cr
ater
lake
s)A
ngol
a, B
engo
, Cab
ungo
B. g
lobo
sus
LT67
1918
[81]
B. g
lobo
sus
LT67
1947
[81]
Ang
ola,
Ben
go, B
urga
lhei
ra
esco
laB.
glo
bosu
sLT
6719
25 [8
1]
Ang
ola,
Lua
nda,
Can
dim
ba/
Mux
ima
B. g
lobo
sus
LT67
1928
[81]
Ang
ola,
Kw
anza
Nor
te, C
acus
oB.
glo
bosu
sLT
6719
36 [8
1]
Ang
ola,
Lua
nda,
Cas
sefo
B. fo
rska
liiLT
6719
55 [8
1]
Ang
ola,
Lua
nda,
Caq
uila
B. fo
rska
liiLT
6719
56 [8
1]
Ang
ola,
Ben
go, P
orto
man
‑gu
eira
sB.
fors
kalii
LT67
1950
[81]
B. fo
rska
liiLT
6719
51 [8
1]
Ang
ola,
Kw
anza
Nor
te,
N’D
alat
ando
B. fo
rska
liiLT
6719
57 [8
1]
Ang
ola,
Ben
go, I
cau
Wan
doB.
fors
kalii
LT67
1966
[81]
Page 9 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
B. fo
rska
liiLT
6719
49 [8
1]
Ang
ola,
Mal
anje
, Car
lang
aB.
fors
kalii
LT67
1961
[81]
B. fo
rska
liiLT
6719
62 [8
1]
Ang
ola,
Mal
anje
, Cal
andu
laB.
fors
kalii
LT67
1964
[81]
Ang
ola,
Qui
fang
ondo
B. fo
rska
liiA
M28
6306
[30]
Cam
eroo
n, P
epto
noum
‑wes
tC
AM
5.63
306°
N10
.635
28°E
B. tr
opic
us2
CA
M1.
1KJ
1574
95 [5
4]
B. tr
opic
usC
AM
1.2
KJ15
7496
[54]
B. tr
opic
usKJ
1574
97 [5
4]
Burk
ina
Faso
, Mog
tedo
bar
rage
12.3
0647
°N−
0.82
783°
EB.
fors
kalii
AM
2863
10 [3
0]
B. g
lobo
sus
AM
2862
93 [3
0]
B. tr
unca
tus
AM
2863
15 [3
0]
Cam
eroo
n, P
epto
noum
‑eas
tB.
trop
icus
KJ15
7492
[54]
B. tr
opic
usKJ
1574
94 [5
4]
Cam
eroo
n, B
erto
ua4.
5888
9°N
13.6
8111
°EB.
trun
catu
sKJ
1352
87 [5
4]
Cam
eroo
n, M
ourt
ous
B. g
lobo
sus
KJ15
7471
[54]
Cam
eroo
n, Y
agou
aB.
glo
bosu
sKJ
1574
72 [5
4]
Cam
eroo
n, K
apris
siB.
glo
bosu
sKJ
1574
75 [5
4]
Cam
eroo
n, G
ouno
ugou
B. g
lobo
sus
KJ15
7473
[54]
Cam
eroo
n, O
urou
douk
oudj
eB.
glo
bosu
sKJ
1574
74 [5
4]
Cam
eroo
n, K
aele
B. se
nega
lens
isKJ
1574
81 [5
4]
B. se
nega
lens
isKJ
1574
80 [5
4]
Cam
eroo
n, K
ekem
B. tr
unca
tus
KJ13
5289
[54]
Cam
eroo
n, M
okol
oB.
trun
catu
sKJ
1352
91 [5
4]
Cam
eroo
n, L
oum
B. tr
unca
tus
KJ13
5295
[54]
DR
Cong
o, L
ake
Kivu
B. tr
unca
tus
HQ
1215
61 [3
2]
Egyp
t, Q
uena
26.1
7306
°N32
.166
11°E
B. tr
unca
tus
KJ13
5304
[54]
Giz
a, E
gypt
30.1
4139
°N31
.076
94°E
B. tr
unca
tus
KJ13
5300
[54]
Iran,
Kho
uzes
tan
B. tr
unca
tus
KT36
5867
Keny
a, N
imbo
dze
B. n
asut
us
nasu
tus
AM
9218
41 [3
0]
Keny
a, K
isum
u, K
anda
ria d
amB.
glo
bosu
sA
M28
6286
[30]
Keny
a, K
inan
goB.
glo
bosu
sA
M92
1844
[30]
Keny
a, K
ache
tuB.
glo
bosu
sA
M92
1847
[30]
Page 10 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
Keny
a, M
wam
duli
B. g
lobo
sus
AM
9218
50 [3
0]
Mal
awi,
Lake
Mal
awi
LMB.
nya
ssan
us1
LM1.
1A
M92
1838
[30]
B. a
frica
nus
AM
2862
96 [3
0]
Nig
er, S
aton
iB.
fors
kalii
AM
2863
08 [3
0]
B. tr
unca
tus
AM
2863
16 [3
0]
Nig
er, T
onid
aB.
glo
bosu
sA
M28
6294
[30]
B. g
lobo
sus
AM
9218
08 [3
0]
Nig
eria
, Iba
ro, O
gun
7.15
000°
N3.
1166
7°E
B. tr
unca
tus
FN54
6781
[80]
Nig
eria
, Ilie
8.25
000°
N4.
9666
7°E
B. tr
unca
tus
FN54
6797
[80]
B. tr
unca
tus
FN54
6797
[80]
Nig
eria
, Osh
ogbo
8.08
333°
N4.
6666
7°E
B. tr
unca
tus
FN54
6805
[80]
B. tr
unca
tus
FN54
6805
[80]
Nig
eria
, Oju
Ala
roB.
glo
bosu
sFN
5468
15 [8
0]
Nig
eria
, Ipo
gun
B. g
lobo
sus
FN54
6814
[80]
Nig
eria
, Im
ala,
Odo
B. tr
unca
tus
FN54
6787
[80]
Nig
eria
, Aw
uru
B. tr
unca
tus
FN54
6786
[80]
Om
anB.
wrig
hti
AM
2863
18 [3
0]
Rwan
da, r
ice
sche
me
dam
lake
−1.
2865
2°N
30.3
1509
°E13
49Bu
linus
sp.
UG
SB 1
6778
2253
2M
N55
1579
B. tr
unca
tus
UG
SB 1
6777
2253
1M
N55
1578
Rwan
da, L
ake
Muh
azi
−1.
8591
2°N
30.4
9039
°E14
52B.
trun
catu
sU
GSB
167
5522
511
MN
5515
81
Rwan
da, L
ake
Muh
azi
−1.
8484
3°N
30.4
7826
°E14
55B.
trun
catu
sU
GSB
493
622
549
MN
5515
80
Sard
inia
, Pos
ada
40.6
3487
°N9.
6753
7°E
B. tr
unca
tus
AM
2863
12 [3
0]
Sao
Tom
e an
d Pr
inci
pe, S
ao
Tom
e Is
land
B. fo
rska
liiA
M28
6305
[30]
Sene
gal,
Dak
arB.
fors
kalii
AM
2863
07 [3
0]
Sene
gal,
Dia
ma
15.4
3611
°N16
.233
89°E
B. tr
unca
tus
KJ13
5306
[54]
Sene
gal, T
ouka
rB.
sene
gale
nsis
KJ15
7483
[54]
Sene
gal,
Dio
hine
B. se
nega
lens
isKJ
1574
84 [5
4]
B. se
nega
lens
isKJ
1574
85 [5
4]
Sene
gal,
Poud
aye
B. se
nega
lens
isKJ
1574
86 [5
4]
Sout
h A
frica
, Lak
e Si
baya
SAB.
nat
alen
sis1
SA1.
1A
M28
6311
[30]
Sout
h A
frica
, Pie
term
aritz
burg
B. g
lobo
sus
AM
2862
89 [3
0]
B. g
lobo
sus
AM
2862
90 [3
0]
Sout
h A
frica
, Dur
ban
Isip
ingo
B. a
frica
nus
AM
2862
95 [3
0]
Page 11 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
Tanz
ania
, Muy
uni,
Ung
uja
−6.
3784
5°N
39.4
6415
°EB.
nas
utus
pr
oduc
tus
AM
2862
99 [3
0]
Tanz
ania
, Njo
mbe
Kib
ena
TZ−
9.20
382°
N34
.784
02°E
B. tr
opic
us1
TZ1.
1A
M92
1834
[30]
B. tr
opic
usTZ
1.2
AM
9218
37 [3
0]
B. tr
opic
usTZ
1.3
AM
9218
42 [3
0]
Tanz
ania
, Lak
e Sa
gara
,−
5.25
140°
N31
.085
18°E
Bulin
us s
p.A
M28
6298
[30]
Tanz
ania
, Iha
yabu
yaga
B. n
asut
us p
ro-
duct
usA
M28
6300
[30]
B. n
asut
us
prod
uctu
sA
M28
6301
[30]
Tanz
ania
, Njo
mbe
Ruj
ewa
B. n
asut
us
prod
uctu
sA
M92
1833
[30]
Tanz
ania
, Zan
ziba
r Vito
nguj
i, Pe
mba
Isla
nd−
5.23
378°
N39
.828
43°E
B. n
asut
us
prod
uctu
sA
M92
1809
[30]
Tanz
ania
, Zan
ziba
r, Pe
mba
Is
land
−5.
1712
0°N
39.8
2198
°EB.
nas
utus
pr
oduc
tus
AM
9218
11 [3
0]
Tanz
ania
, Zan
ziba
r, Pe
mba
Is
land
−4.
9280
3°N
39.7
3785
°EBu
linus
sp.
AM
9218
32 [3
0]
Tanz
ania
, Zan
ziba
r, Pe
mba
Is
land
B. g
lobo
sus
MH
0140
41 [8
2]
Tanz
ania
, Zan
ziba
r, Pe
mba
Is
land
, Kan
gaga
niB.
bar
thi
AM
9218
18 [3
0]
Tanz
ania
, Zan
ziba
r, M
afia
Isla
ndB.
nas
utus
na
sutu
sA
M92
1831
[30]
Tanz
ania
, Zan
ziba
r, M
afia
Isla
nd,
Kang
a sw
amp
B. b
arth
iA
M92
1814
[30]
B. g
lobo
sus
AM
9218
23 [3
0]
Tanz
ania
, Irin
gaB.
glo
bosu
sA
M28
6288
[30]
B. g
lobo
sus
AM
9218
21 [3
0]
Tanz
ania
, Ung
unja
, Kin
yasi
niB.
glo
bosu
sA
M92
1839
[30]
Uga
nda,
Lak
e Vi
ctor
iaLV
−0.
3037
1°N
32.2
8927
°E12
28B.
trop
icus
1LV
1.1
UG
SB 1
6774
2253
0M
N55
1506
Uga
nda,
Lak
e G
eorg
eLG
B. fo
rska
lii2
LG1.
1H
Q12
1586
[32]
B. fo
rska
liiLG
1.2
HQ
1215
85 [3
2]
Uga
nda,
Lak
e Ed
war
dLE
B. fo
rska
lii2
LE1.
1H
Q12
1583
[32]
B. fo
rska
liiLE
1.2
HQ
1215
84 [3
2]
Uga
nda,
Lak
e A
lber
tLA
B. fo
rska
lii4
LA1.
1H
Q12
1582
[32]
Page 12 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Tabl
e 1
(con
tinu
ed)
Cra
ter fi
eld
Loca
tion
Lake
cod
eCo
ordi
nate
sA
ltitu
de
(m)
Spec
ies
No.
of
hapl
otyp
esa
Spec
imen
co
dea
Spec
imen
vo
uche
rPr
ep.
no.
Gen
Bank
ID
[refe
renc
e]La
titud
eLo
ngitu
de
B. fo
rska
liiLA
1.2
HQ
1215
80 [3
2]
B. fo
rska
liiLA
1.3
HQ
1215
81 [3
2]
B. fo
rska
liiLA
1.4
HQ
1215
87 [3
2]
B. tr
opic
us2
LA1.
5H
Q12
1564
[32]
B. tr
opic
usLA
1.6
HQ
1215
65 [3
2]
B. tr
opic
usLA
1.7
GU
1767
51 [5
0]
B. tr
opic
usLA
1.8
GU
1767
50 [5
0]
Uga
nda,
Too
nya,
Lak
e A
lber
tB.
trun
catu
sG
U17
6749
[50]
Uga
nda,
Boo
ma,
Lak
e A
lber
tB.
trun
catu
sG
U17
6747
[50]
Uga
nda,
Kat
osho
sw
amp
KSB.
fors
kalii
2KS
1.1
HQ
1215
88 [3
2]
B. fo
rska
liiKS
1.2
HQ
1215
87 [3
2]
B. tr
unca
tus
HQ
1215
63 [3
2]
B. tr
unca
tus
HQ
1215
62 [3
2]
Uga
nda,
Lak
e Ta
ngan
yika
LTB.
fors
kalii
3LT
1.1
HQ
1215
90 [3
2]
B. fo
rska
liiLT
1.2
HQ
1215
89 [3
2]
B. fo
rska
liiLT
1.3
HQ
1215
87 [3
2]
Uga
nda,
Alb
ert N
ile R
iver
3.47
032°
N31
.922
67°E
B. g
lobo
sus
AM
2862
91 [3
0]
Uga
nda,
Lak
e Ky
oga
1.82
235°
N33
.537
25°E
B. n
asut
us
prod
uctu
sA
M92
1815
[30]
Bulin
us s
p.A
M92
1819
[30]
Uga
nda,
Kah
irim
bi−
0820
5° N
30.8
559
°E12
49Bu
linus
sp.
UG
SB 2
4296
1941
5M
N55
1577
Uga
nda,
Mar
amag
ambo
For
est
Bulin
us s
p.H
Q12
1591
[32]
Uga
nda,
Lak
e Vi
ctor
ia0.
1370
7°N
33.6
0149
°E11
35B.
trun
catu
sU
GSB
167
5722
513
MN
5515
82
B. tr
unca
tus
UG
SB 1
6758
2251
4M
N55
1583
Uga
nda,
Nile
Riv
er1.
6924
9°N
32.0
9664
°E10
35B.
trun
catu
sU
GSB
167
6022
516
MN
5515
85
B. tr
unca
tus
UG
SB 1
6759
2251
5M
N55
1584
Zim
babw
e, la
bora
tory
str
ain
ZWB.
trop
icus
1ZW
1.1
AY28
2583
[37]
Not
es: D
ata
incl
ude
the
crat
er fi
eld
regi
on, g
eogr
aphi
cal c
oord
inat
es, a
ltitu
de (o
btai
ned
from
Goo
gleE
arth
Pro
1.0
.0.1
), nu
mbe
r of h
aplo
type
s, sp
ecim
en c
ode
(use
d fo
r the
net
wor
k an
alys
es),
spec
ies,
DN
A p
repa
ratio
n nu
mbe
r (pr
ep. n
o.),
spec
imen
vou
cher
(UG
SB, U
nive
rsity
of G
iess
en S
yste
mat
ics
and
Biod
iver
sity
col
lect
ion)
and
Gen
Bank
acc
essi
on n
umbe
r. Bu
linus
spe
cim
ens
from
Lak
e Ky
anin
ga c
ould
not
be
ampl
ified
. Not
e th
at a
few
G
enBa
nk n
umbe
rs o
f oth
er a
utho
rs a
re o
ccur
ring
in m
ore
than
one
loca
lity
beca
use
they
repr
esen
t sam
e ha
plot
ypes
. Loc
ality
info
rmat
ion
and
geog
raph
ical
coo
rdin
ates
of G
enBa
nk s
eque
nces
are
pro
vide
d as
they
wer
e pu
blis
hed
a Num
ber o
f hap
loty
pes
and
spec
imen
cod
es a
re o
nly
give
n fo
r spe
cim
ens
used
in th
e ne
twor
k an
alys
esb L
akes
that
did
not
yie
ld p
opul
atio
ns o
f Bul
inus
c Cra
ter l
ake
whe
re c
urre
ntly
no
Bulin
us w
ere
foun
d bu
t seq
uenc
es o
n G
enBa
nk w
ere
avai
labl
e
Page 13 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Alignment and dataset compositionThe study comprised specimens from all crater lakes where Bulinus occurred. Furthermore, additional speci-mens from surrounding watershed and other major aquatic systems were included in order to better trace regional affinities. These are samples from a rice scheme and lakes Muhazi (Rwanda), Mburo, Victoria and the Nile River system in Uganda. In total, material from 43 sampling localities and 85 specimens was used for DNA analyses. Sequences were edited and aligned in BioEdit version 7.0.5 [41]. All 84 newly generated sequences were Blast-searched against the GenBank sequence database. The newly generated sequences were supplemented with all previously published relevant sequences on GenBank. The resulting dataset was aligned using the ClustalW multiple alignment tool in BioEdit after removing redun-dant haplotypes.
Phylogenetic and phylogeographical analysesBayesian inference analysis was based on a total of 152 sequences (unique haplotypes) originating from our new sampling, as well as from GenBank data. The analysis was performed utilizing MrBayes version 3.2.2 [42] using the following settings: ngen = 4,000,000, samplefreq = 200, ‛burn-in’ = 5000 (25%) and HKY+I+Γ as the best-fit sub-stitution model (selected using jModelTest version 2.1.4 [43] under the AIC, AICc and BIC criteria). The effective sample size (ESS) values were examined in Tracer ver-sion 1.5.0 [44] indicating for all major parameters val-ues > 800. Statistical parsimony network analyses for all major clades found to contain crater lake specimens were conducted using TCS version 1.21 [45]. The sub-datasets were selected according to the results of the phylogenetic analysis, two specific clades were selected: Clade 1 and 2). The connection limit was set to either 95% (Clade 1) or 90% (Clade 2).
Genetic diversityThe final cluster analysis of crater lake similarity was based on presence/absence of 31 haplotypes of Bulinus, the Bray-Curtis similarity measure was used (PAST ver-sion 3.22) [46].
The relationship of altitude and distribution of hap-lotype diversity across the crater lake fields was tested
using correlation analysis implemented in PAST version 3.22 [46].
ResultsHost species identification and diversityOut of 58 crater lakes sampled, Bulinus spp. snails were found in 34 belonging to the two crater fields Bunyaru-guru and Ndali-Kasenda. Although Bulinus spp. snails were sampled in Lake Kyaninga (Fort Portal), the sam-ples did not yield any DNA for analysis due to techni-cal issues. However, there is a high likelihood that the Bulinus spp. from this lake belong to one of the impor-tant S. haematobium hosts, Bulinus globosus, based on the shell shape (Additional file 1: Figure S1c). Lakes Rwijongo (Bunyaruguru), Mubiro and Kanyabutetere (Ndali-Kasenda) yielded Bulinus spp. shells only during the sampling. For Lake Rwijongo, sequences from the GenBank database were available and were included in the analyses. The rest of the crater lakes had either other gastropods or no snails at all. Bulinus spp. co-occurred with Biomphalaria spp. in 28 lakes. Three species of Buli-nus were found, i.e. B. forskalii, B. truncatus and B. tropi-cus. The first was found in only two crater lakes (Mirambi and Kibungo), which are in close proximity located in the Ndali-Kasenda crater field. Bulinus truncatus exclusively occurred in Lake Kyasanduka in the Maramagambo For-est (Bunyaruguru). Bulinus tropicus was dominant, found in the rest of the crater lakes that harbored Bulinus spp. (Table 1). In addition, neither B. forskalii nor B. truncatus occurred sympatrically with B. tropicus. All the three B. forskalii and the four B. truncatus specimens genotyped showed the same haplotypes. Bulinus tropicus portrayed a high variability within and from one lake to another across the crater lakes fields. In total, this study is com-posed of 33 haplotypes in 34 crater lakes (one haplotype for B. forskalii and B. truncatus and 31 for B. tropicus).
Phylogenetic relationships and biogeographical affinitiesThe Bayesian inference analysis showed that Bulinus specimens genotyped for this study are distributed across three of the four Bulinus species groups/complex, spe-cifically the B. forskalii and B. africanus groups and the B. truncatus/B. tropicus complex (Fig. 3). Bulinus tropi-cus from the crater lakes clustered exclusively within a
(See figure on next page.)Fig. 3 Bayesian inference phylogenetic tree for Bulinus spp. based on cox1 gene sequences. Specimens are given with locality information as to country of origin and localities in some cases. The DNA preparation numbers are provided. Crater lake names are provided and the two specific clades of B. forskalii (Clade 1) and B. tropicus (Clade 2) are highlighted with light grey boxes. Crater lake populations are represented at the end of the branch by red boxes, while regional and non‑regional (= others) populations are demonstrated by green and grey boxes, respectively. The outgroup Indoplanorbis exustus is not shown. The tree has been partly graphically collapsed (for the full tree see Additional file 2: Figure S2). Bayesian posterior probabilities (pp) are given for deeper nodes (when pp ≥ 0.95). The scale‑bar represents substitutions per site according to the model of sequence evolution. The number of individuals per haplotype is shown in parentheses for the two specific clades (for details see Figs. 4, 5)
Page 14 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
highly supported B. tropicus clade (Clade 2, pp = 0.97, see Fig. 3 and Additional file 2: Figure S2) of the B. tropicus/B. truncatus complex (pp = 1.00). However, B.
tropicus specimens from the crater lakes did not form a monophyletic group. Instead, the clade comprising the crater lake samples also included B. tropicus from
B. forskalii, Lake Mirambi, UG )955155NM( )4(B. forskalii, Lake Edward, UG (HQ121583)B. forskalii, Lake Albert, UG (HQ121582)
B. forskalii, Lake Edward, UG (HQ121584)B. forskalii, )785121QH( )3(ZT,pmawsahsotaK
B. forskalii, TZ (HQ121588)
B. forskalii, TZ (HQ121589)
B. forskalii, Lake Albert, UG (HQ121580)
B. forskalii, Lake George, UG (HQ121585)
B. forskalii, SN (AM286307)B. camerunensis, CM
B. forskalii, NE (AM286308)B. forskalii, BF (AM286310)
Bulinus sp., dam lake, RW (MN551579)Bulinus sp., Maramagambo Forest, UG (HQ121591)
Bulinus sp., TZ (AM921832)B. barthi, TZ
B. senegalensis, CM,SNBulinus sp., Lake Kyoga, UG (AM921819)B. tropicus, Lake Kyanga, UG )335155NM( )7(B. tropicus, Lake Nyamuteza, UG (MN551546)
B. tropicus, Lake Rwenjuba, UG (MN551549)B. tropicus, Lake Njarayabana, UG (MN551568)
B. tropicus, Lake Rwenjuba, UG (MN551548)
B. tropicus, Lake Nyamugosani, UG )055155NM( )4(B. tropicus, Lake Nyinambuga, UG )155155NM( )6(
B. tropicus, Lake Mwengenyi, UG (MN551558)
B. tropicus, Lake Nyinabulita, UG (MN551530)
B. tropicus, Lake Nkuruba, UG (HQ121569)B. tropicus, Lake Nyinabulita, UG (MN551531)B. tropicus, Lake Nyamirima, UG )345155NM( )5(B. tropicus, Lake Nkuruba, UG (HQ121566)
B. tropicus, Lake Wankenzi, UG (MN551505)B. tropicus, Lake Wankenzi, UG (MN551507)B. tropicus, Lake Wankenzi, UG (MN551509)B. tropicus, Lake Nyamugosani, UG )225155NM( )9(B. tropicus, Lake Kasenda, UG )425155NM( )01(B. tropicus, Lake Lugembe, UG )065155NM( )2(B. tropicus, Lake Nyinambuga, UG (HQ121573)
B. tropicus, Lake Victoria, UG (MN551506)
B. tropicus, )057671UG( )2(GU,treblAekaLB. tropicus, )465121QH( )2(GU,treblAekaL
B. tropicus, Lake Mafuro, UG )015155NM( )5(B. tropicus, Lake Kyamwiga, UG )615155NM( )4(
B. tropicus, )438129MA( )3(ZT,anebiKebmojNB. tropicus, Lake Kanyamukali, UG )305155NM( )5(B. tropicus, Lake Kitere, UG (MN551541)B. tropicus, Lake Nyabikere, UG )825155NM( )3(
B. tropicus, )675121QH( )2(GU,ewgnaynaKekaLB. tropicus, Lake Mafuro, UG )115155NM( )6(B. tropicus, Lake Kanyamukali, UG )625155NM( )3(B. tropicus, Lake Rwandakara, UG (MN551562)B. tropicus, Lake Kasenda, UG (HQ121579)B. nyassanus, Lake Malawi, UG (AM921838)
B. natalensis, Lake Sibaya, ZA (AM286311)B. tropicus, ZW (AY282583)
B. truncatus, Lake Muhazi, RW (MN551581)B. truncatus, dam lake, RW (MN551578)B. truncatus, Lake Muhazi, RW 085155NM )(
B. truncatus, TZ (HQ121562)B. truncatus, TZ (HQ121563)
B. truncatus, BF,NE,NGB. truncatus, Lake Victoria, UG (MN551582)B. truncatus, Lake Victoria, UG (MN551583)B. truncatus, Nile River, UG (MN551585)
B. truncatus, Lake Albert, UG (GU176749)
B. truncatus, CM,EG,NG,IR
B. truncatus, IT (AM286312)B. truncatus, Nile River, UG (MN551584)B. truncatus, NG (FN546781)B. truncatus, 747671UG )(GU,treblAekaL
B. truncatus, CD (HQ121561)B. truncatus, CM (KJ135287)
B. truncatus, SN (KJ135306)B. tropicus, CM
B. wrighti, IR (AM286318)
B. africanus groupB. forskalii groupB. truncatus/tropicus complexB. reticulatus group
A
Regional population Crater lake population
Others
BCD
Clade 2
Clade 1
B. globosus, AO,BF,CM,NE,NG,SN,ZA
B. globosus, KE,TZB. globosus, )192682MA(GU,reviReliNtreblA
B. globosus, TZ,ZAB. globosus, KE,TZ
Bulinus sp., TZ (AM286298)B. nasutus productus, TZB. nasutus productus, TZ (AM286300)B. nasutus productus, Lake Kyoga, UG (AM921815)Bulinus sp., Kahirimbi, UG (MN551577)
B. nasutus nasutus, KE,TZB. nasutus productus, TZ (AM286299)B. nasutus productus, TZ (AM921809)B. nasutus productus, TZ (AM921811)
2
B. forskalii, AO,ST
2
17
3
4
4
2
2
B. forskalii, Lake Tanganyika, UG (HQ121590)
B. forskalii, Lake Albert, UG (HQ121581)
12
26
B. tropicus, Lake Mafuro, UG (HQ121571)
9
B. truncatus, Lake Kyasanduka, UG 575155NM )(
4
A
B
C
D
0.31.00
0.980.99
1.00
1.00
0.96
1.00
1.00
1.00
0.99
0.961.00
1.00
0.95
1.00
1.00
0.97
1.00
1.00
Page 15 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
Lake Victoria (MN551506), Lake Albert (GenBank: HQ121564, GU176750) and Njombe Kibena in Tan-zania (GenBank: AM921834). Specimens from Lake Malawi (GenBank: AM921838), South Africa (Gen-Bank: AM286311) and a laboratory strain from Zimba-bwe (GenBank: AY282583) clustered in a basal position to the B. tropicus clade that comprised the crater lake samples. The specimen from Lake Kyasanduka and some specimens of regional populations clustered within the B. truncatus assemblage. These populations derived from Lake Victoria, Nile River, Lake Muhazi and other places in Rwanda, which are in close proximity to the crater field systems in western Uganda (Figs. 1, 3). The B. trun-catus assemblage also includes populations from loca-tions as far away as Nigeria, Cameroon, or Egypt and Burkina Faso.
The B. forskalii group was genetically very diverse as evidenced by the branch lengths variation with the two crater lake populations clustering with other Ugandan populations from lakes Albert, Edward and George. A distinct and highly supported clade (clade 1, pp = 1.00, see Fig. 3) also comprised populations from the Kato-sho swamp (Tanzania) and Lake Tanganyika (Tanzania). A Bulinus sp. from a dam lake connected to a rice field irrigation system in Rwanda (MN551579) belonged to the Bulinus forskalii group but clustered in a different subclade. It also included another Bulinus sp. from Mara-magambo Forest, Uganda (GenBank: HQ121591), a place not far from the crater lake fields. Yet another Bulinus sp. from Lake Kyoga clustered with the B. senegalensis sub-clade of the B. forskalii group.
Another Bulinus sp. (MN551577) from Kahirimbi in Lake Mburo/Nakivale wetland system, about 120 km south of the crater lakes, belonged the B. africanus spe-cies group (Fig. 3). It was part of a clade that comprised B. nasutus from Lake Kyoga, Uganda and other regions in Tanzania. Bulinus globosus from Nile River (Moyo, Uganda) was the geographically closest occurrence of this species to the crater lakes in our dataset. Both reso-lution and support values were low within B. tropicus and B. forskalii clades. The phylogeographical structure for those clades were thus specifically analysed with a parsi-mony network analysis.
Phylogeographical patternsBulinus forskalii from the crater lakes formed a single network with GenBank haplotypes from the surrounding lakes, i.e. Lake Albert in the north, Lake Edward in the west and Lake George in the east. A few haplotypes from regions outside Uganda, such as Lake Tanganyika and nearby Katosho swamp also appeared in the network, whereas others from Rwanda or the Maramagambo For-est east of Lake Edward did not (Fig. 4). All the three
crater lake specimens represented one haplotype and together with a haplotype (GenBank: HQ121586) from Lake George formed the most probable ancestral haplo-type for the network. Haplotypes from far away regions were also reconstructed as distantly related. For exam-ple, there were 11 and 10 mutational steps between hap-lotypes from Lake Tanganyika (GenBank: HQ121590, HQ121589). On the other hand, haplotypes from nearby regions such as Lake Edward and Lake George were reconstructed either as relatively similar (e.g. Gen-Bank: HQ21582, HQ21583, HQ121586) or as relatively far distant in terms of mutational steps (e.g. GenBank: HQ121580).
The single haplotype network of B. tropicus based on a 90% connection limit contained 38 haplotypes (Fig. 5). Haplotype 11 (Fig. 5) was present in six lakes in Ndali-Kasenda was reconstructed as the most probable ances-tral haplotype. The haplotypes of both the Ndali-Kasenda and the Bunyaruguru crater fields were very diverse
Others
Ndali-Kasenda crater lakes
Regional population
LA1.2
LA1.3
LA1.1
LG1.2 LE1.1
LT1.1
4
LT1.2
KS1.2,
LA1.4,LT1.3
LE1.2
KS1.1
KIG1.1,
LG1.1,MRB1.1-2
Fig. 4 Statistical parsimony network of cox1 sequences for Bulinus forskalii. The network corresponds to the B. forskalii clade highlighted in Fig. 3. The connecting limit was 95%. The Ndali‑Kasenda crater lake specimens are indicated in red. Haplotypes originating from regional and non‑regional (= others) are in green and grey, respectively. The most probable ancestral haplotype is indicated by a bold circle
Page 16 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
ZW1.1
WKZ1.1
WKZ1.2
WKZ1.3
NBT1.2NRB2.3
NNG2.1
LM1.1
KSD1.2,
NKR2.2,
NRB1.1,
MRR1.1-2,
NGS1.1-2,
NSG1.1-2
NMM1.1-2,
NRB1.3-4,
NRB2.2
TZ1.1-3LA1.6,
LA1.8
LA1.5,
LA1.7
LV1.1
MFR1.1,
MRM1.1-2,
NMM2.1,
NRB2.1
SA1.1
KMG1.1-2,
BGJ1.1-2
NKR1.1-2,
KSD2.2KAG2.1,
CAM1.1
RKR1.1KSD2.3
KTR1.1
KML1.3-5,
KTR1.2,
KYG1.1
LGB1.1-2
NRB2.4
NBT1.1
RJB1.2MGY1.2
NJN1.2
RJB1.1
KAG1.2,
NGS1.3,
NKR2.1,
NJN1.1
NTZ1.2
KAG1.1,
KTD1.1-2, KYG1.2,
MBA1.1-2,
NTZ1.1
MFR2.1,
NGU2.1,
RWG2.3
CAM1.2,
NNG1.1/4,
MBA1.3,
MGY1.1,
RWG2.2
KGZ1.1-2,
KSD2.1,
MFR1.2,
NGU1.1-2
OthersBunyaruguru crater lakes
Ndali-Kasenda crater lakes Regional population
KSD1.1, MBA2.1,
NHR1.1-2,NNG1.2-3,
NRB1.2 , RKZ1.1-3
KML1.1-2,
RKR1.2
Fig. 5 Statistical parsimony network of cox1 sequences for Bulinus tropicus. The network corresponds to the B. tropicus clade highlighted in Fig. 3. The connecting limit was 90%. The two crater lakes fields of Ndali‑Kasenda and Bunyaruguru are coloured red and light red, respectively. Haplotypes connected but occurring in other systems are represented in green and grey for regional and non‑regional (= others) populations respectively. The most probable ancestral haplotype is indicated by a bold circle
Page 17 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
ranging from a few or no mutational steps, to as many as 17.
In most cases, haplotypes of the Ndali-Kasenda crater field were unique with a few exceptions where a haplo-type was shared with either samples from the Bunyaru-guru crater field (haplotypes 7 and 8 in Fig. 5), outside the region (haplotype 32 in Fig. 5) or both (haplotype 6 in Fig. 5). Lakes Kyamwiga and Bugwagyi of the Bunyaru-guru crater field had exclusive haplotypes. Some lakes had quite distantly genetically related haplotypes such as MN551511, MN551510 and HQ121571, all of which are from Lake Mafuro located in the Bunyaruguru crater field. A haplotype from as far as Cameroon was identical to three crater lake samples of the Ndali-Kasenda crater field that are in close proximity to one another and Lake Rwijongo of the Bunyaruguru crater field (haplotype 6 in Fig. 5). Except for the Cameroonian, all haplotypes from outside crater lake systems are unique. These are haplotypes from Lake Victoria, Lake Albert and Tanza-nia. They belonged to a single group connected to crater lake haplotypes by a minimum of four mutation steps to the Ndali-Kasenda haplotypes via a Tanzanian haplo-type (haplotype 37 in Fig. 5), Lake Albert (haplotype 35
in Fig. 5) and five mutation steps to a Bunyaruguru hap-lotype via a Lake Victoria haplotype (haplotype 34 in Fig. 5). Three haplotypes were extremely distant from the core network, i.e. samples from South Africa (haplotype 26 in Fig. 5), B. nyassanus from Lake Malawi (haplotype 38 in Fig. 5) and the laboratory strain from Zimbabwe (haplotype 10 in Fig. 5).
Genetic diversityThe cluster analysis based on the presence/absence of haplotypes did not result in a clear pattern (Fig. 6). Whereas some lakes clustered together, others remained unclustered. Lakes Nyungu (NGU), Kigezi (KGZ) and Mafuro (MFR) all belong to the Bunyaruguru crater field cluster together. Other lakes belonging to that cra-ter field such as lakes Bugwagyi (BGJ) Kyamwiga (KMG) were clustered too, whereas Rwijongo (RWG) did not cluster together with any of the two groups in the same crater field. The numerous lakes of the Ndali-Kasenda field formed three main clusters. Lakes Wankenzi (WKZ), Lugembe (LGB), Rwenjuba (RJB) and Nyinabu-litwa (NBT) did not cluster to any of the other groups. Some lakes in the two crater fields tended to cluster
WKZ
RJB
NBT
LGB
KMG
BGJ
KSD
MRR
NSG
NKR
NGSNJN M
RMNM
MNRBM
FRNGUKG
ZM
BANN
GNH
RRK
ZM
GYRW
GKA
GKY
GKT
DNT
ZRKRKM
LKT
R
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Sim
ilarit
y
Ndali-Kasenda crater lakes Bunyaruguru crater lakes
Fig. 6 Cluster analysis of crater lake similarity based on presence/absence of 31 haplotypes of Bulinus tropicus. The Bray‑Curtis similarity measure was used. Three letter codes refer to the respective lakes in Table 1. The haplotype matrix is given in Additional file 3: Table S1
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together, for example Lake Nyamirima (NMM) with Lake Murambi (MRM) and Lake Rwijongo (RWG) with Lake Mwengenyi (MGY). Lakes that came out to be more sim-ilar according to this analysis, for example lakes Nyahira (NHR) and Rukwanzi (RKZ), or lakes Nyanswiga (NSG) and Muligamire (MRR) are not geographically related.
Haplotype diversity across the crater lakes’ altitudinal gradientThe distribution of haplotype diversity along the altitude gradient was unimodal (Fig. 7). A high haplotype diver-sity was realized in an altitude range between 1200 to 1300 m a.s.l., with 16 different haplotypes. Lake Nkuruba with the most haplotype diversity and situated at an alti-tude of 1517 m a.s.l) was represented by eight specimens and six unique haplotypes. The lakes in the Bunyaruguru crater field, which are at the lowest altitudes exhibited limited haplotype diversity (n = 6). Based on the present dataset, the haplotype diversity was not correlated with altitude (r = 0.26813, P = 0.12787).
DiscussionBulinus species in the crater lakesTo date, historical records provide very restricted infor-mation on molluscs in crater lakes in Uganda [33]. Although Bulinus tropicus has been present there for quite a while already, not much more was known hith-erto, except for the study by Nalugwa et al. [32] on Bulinus species complexes in the Albertine Rift, which included a few crater lakes of western Uganda. In this study, Bulinus tropicus by far dominates in the crater lakes, whereas B. forskalii was exclusively found in lakes
of Mirambi and Kibungo that are in close proximity. Buli-nus forskalii is essentially an Afrotropical species that often occurs in small and even temporary water bodies [26]. It seems less common in colder climates of high-lands in eastern Africa and has been found up to 1800 m a.s.l. in Ethiopia [47]. Bulinus forskalii is not known to be naturally infected with S. haematobium (see [48]). Bulinus tropicus is a widespread species in eastern and southern Africa, but unlike its sibling species B. trunca-tus, is apparently not acting as intermediate host for S. haematobium, the parasite causing urogenital schistoso-miasis [26, 27]. Bulinus tropicus is known to occur up to 2700 m a.s.l. in Kenya [26]. This species is extremely flex-ible ecologically, i.e. it exhibits a high tolerance towards cooling and drought conditions and even to only tempo-rary availability of habitat. Such conditions might exist in the crater lakes of western Uganda, where extensive lake level fluctuations over seasonal periods have been docu-mented ([49]; Tumwebaze, own observations from his-torical satellite images).
This ecological flexibility might be linked to another very interesting finding of the present study, which is the extremely high diversity of haplotypes of B. tropicus in the crater lakes. It almost matches the total range of genetic diversity hitherto known for this species [30]. Given the fact that the present study was not designed as a population study, the real variability might be even higher.
The fact that so far, the majority of the studied Bulinus spp. populations belonged to B. tropicus, does not mean S. haematobium strains would not occur in the entire crater lakes region. The sibling tetraploid species B. trun-catus, a major intermediate host for S. haematobium in many regions of Africa, has been found in various places along the Albertine Rift [32, 50] and our dataset included populations from nearby areas in Uganda and Rwanda (Fig. 3). Although we found it exclusively in one crater lake, sympatric occurrences of the two species are possi-ble [32]. A presence of tetraploid B. truncatus can there-fore not be ruled out without such detailed molecular surveys such as the ones conducted in the present study. The recurrent almost complete absence from the crater lakes of B. truncatus, an ecologically largely flexible spe-cies with high colonizing capacity [26], remains some-what enigmatic. This species has been found confined to altitudes of 2100 m a.s.l., rarely up to 2400 m a.s.l. in Ethiopia [26]. It was present in our study in Lake Victoria, the Nile River and Lake Muhazi, Rwanda, all locations in a radius of just c.250 km. Bulinus globosus, another potential host species, is known from the Nile River, Moyo Province, Uganda [30]. We found another B. nas-utus-like species at the Lake Mburo-Nakivale system in southwestern Uganda. B. nasustus has also been found in
14
10
18
16
12
Haplotype diversityNumber of lakes
1000–1100
Altitudinal range (m a.s.l.)
1100–1200 1200–1300 1300–1400 1400–1500 1500–1600
8
6
4
2
0
Fig. 7 Haplotype diversity versus increase in altitude according to 100 m altitudinal bands for 31 unique haplotypes in 31 crater lakes
Page 19 of 23Tumwebaze et al. Parasites Vectors (2019) 12:565
previous work in Lake Kyoga [30] (Fig. 3). It is therefore perhaps just a matter of time and or chance until other species of Bulinus acting as intermediate hosts for human schistosomiasis are to be found in the crater lakes. The absence of members of the B. africanus group from the crater lakes is noteworthy though. Prediction as to occur-rences of specific snail lineages is difficult since not only time and isolation matter, but also ecology of the small lakes. They are very different from littoral conditions in better studied large lakes such as Albert, Edward and Vic-toria, for which factors favoring a mitigation of the pro-liferation of snail populations have been determined to a much greater extent than in other lotic or lentic natural aquatic systems in East Africa [14, 51–53]. The absence of Bulinus in some of the studied crater lakes might be attributed to limnological conditions [18, 19]. However, 19 lakes where Bulinus was not collected contained other molluscs (Tumwebaze & Albrecht, unpublished data). Repeated sampling during different seasons might help constructing a more complete picture of Bulinus spp. communities in these lakes.
Phylogenetic relationships and biogeographical affinitiesThe phylogenetic study of Bulinus spp. corroborated ear-lier findings that three major species complexes exist in the Albertine Rift region [32], although only B. tropicus was found to be present then in the crater lakes. Our field sampling complements the previously limited knowl-edge of the Bulinus spp. communities of the crater lakes based on the previous effort of Nalugwa et al. [32] on a smaller subset of lakes. The phylogenetic affinities of specimens genotyped from potential source populations, i.e. close geographical proximity, revealed a wide range of genetic variability which is interpreted as reflecting the high morphological and ecological flexibility of the spe-cies, as well as its extraordinary dispersal capacities. The analyses of the B. tropicus subclade (Fig. 3) resulted in few well-supported branches which made tracing a sin-gle origin of the crater lakes populations difficult from a phylogenetic analysis. However, the findings support the hypothesis of highly dynamic fluctuations of popu-lations coming into the crater lakes on a potentially fre-quent basis. It was unexpected to find haplotypes that are known from other places far away in East Africa or even western Africa (Cameroon) in the crater lakes studied here. For the present dataset, the origin of the haplotype network was reconstructed for a haplotype occurring in six lakes of the Ndali-Kasenda lake region, likely reflect-ing the extraordinary diversity in the DNA data. Whereas environmental parameters might account for phenotypic plasticity observed in the B. truncatus/B. tropicus com-plex, no such direct relationships have been shown for the degree of genetic variation. It might be in fact one of
the reasons why B. tropicus is refractory to human-infect-ing schistosomes [26]. Both B. forskalii and B. trunca-tus exhibit less genetic variation on similar geographical scales [25, 54, 55]. Bulinus forskalii from the crater lakes clearly belongs to a very distinct clade of Albertine Rift valley populations and colonization likely happened from nearby sources such as Lakes George, Albert and Edward. Interestingly, this clade also comprises haplotypes from further south, namely Lake Tanganyika. Other species of the B. forskalii group that are geographically closer, such as Lake Kyoga or Rwanda or even the Maramagambo Forest in Queen Elizabeth National Park, Uganda, do represent quite distinct lineages.
Phylogeography and lake patternsSince the crater lakes in western Uganda are roughly 8000 to 10,000 years-old [34], the variation observed is not likely to have developed in that setting given the gen-eral mutation rate of the molecular marker cox1, even if we consider potentially higher rates under tropical condi-tions [56]. It is worth noting that the unique haplotypes often differed by one mutational step only. We must, however, also keep in mind that the coverage of samples of B. tropicus throughout its vast African range is far from being representative and therefore the ‘endemic-ity’ of the unique haplotypes cannot be evaluated with all certainty. The variability in haplotypes might also be reflected in shell shapes as outlined by an example from Lake Mafuro, in which two distinct haplotypes corre-sponded to distinct shell morphs (Additional file 1: Fig-ure S1a, b). The Bulinus snails from Lake Kyaninga have shells that are quite distinct from the rest in the region (Additional file 1: Figure S1c).
One question relates to colonization history, i.e. where the lineages come from. In the case of B. tropicus, our results identified populations from across Africa as potential sources based on genetic affinities, both from nearby source of the Victoria-Nile-Albert system or places considerably far away in Tanzania or even West Africa. The co-occurrence of distantly related haplo-types in a single lake (e.g. Nkuruba or Nyabikere) points towards multiple colonization of the same lake system likely fostered by high propagule pressure. Sharing hap-lotypes regionally hints towards population dispersal by passive means since most of the lakes studied have no hydrological connection. A similar pattern has been found for fishes in the Fort Portal region [57]. Machado-Schiaffino et al. [58] studying the Bunyaruguru crater lakes discovered strong genetic and morphological dif-ferentiation whereby geographically close lakes tend to be genetically more similar. Such a general trend was not obvious when comparing the lakes based on the haplo-type distribution in our study.
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It is important to notice that altitude reflecting cli-mate parameters as earlier predicted [15], did not cor-relate with occurrence and diversity of snail populations. Rather, a more complex interplay of land use, lakes lim-nology, community resistance and stochasticity might account for the presence or absence of certain snail spe-cies and certain haplotypes in the crater lakes.
Parasitological implicationsThis study did not find an immediate risk for urogeni-tal schistosomiasis based on the Bulinus snails identi-fied. However, the identification of up to six partly highly divergent haplotypes in small and young isolated systems such as the crater lakes in Uganda, might hint to either extremely fast evolution or multiple invasions by vectors from various source populations. This involves humans most likely. If this is the case, other species of Bulinus and Biomphalaria might also potentially be introduced. Not only is the probability of the introduction of host snails likely to increase given increased mobility of people in Uganda and international migrations, such as refugees from the crisis regions in surrounding countries, but also are such human flows capable of dispersing non-native parasite strains with them. It should be kept in mind that for example in the neighboring Nile Province of South Sudan prevalence of S. haematobium infection was found to be more than 70% in school children [23] and that the few modelling attempts for urogenital schistosomiasis transmission risk suggest dynamic patterns for the near future [22].
In order to establish an enhanced model of schistoso-miasis prevalence in the crater lakes region, a dedicated survey of infection rates among households adjacent to the lakes that are actually using the water resources of the lakes for various purposes should be conducted. The various ways of how and to what extent water is used directly or indirectly should be assessed and quantified, as the information available with regard to these activi-ties is very limited. The role of human (indirect) trans-port of both host snails and parasites is likely to be more important than previously considered, due to the impor-tant flows of human populations in the region. Move-ment from regions with high infection risk sites around Lake Albert and Lake Victoria or other inland water bodies infested with schistosomiasis might enhance the prevalence of schistosomiasis in the western region of Uganda. There is also need for increased surveillance of new schistosomiasis outbreaks in the crater lakes region especially at higher altitudes in the face of the projected increase in temperature in the near future [59, 60] since crater lakes have shown to be sensitive to climate change [61, 62].
A largely neglected aspect here relates to schistoso-miasis as a disease in livestock. Bulinus tropicus and B. forskalii found in the crater lakes are well-known interme-diate hosts for bovine schistosome species such as S. bovis [63–67]. This parasite is responsible for a large proportion of livestock trematode infections [68], and the parasite’s distribution overlaps largely with S. haematobium. More-over, these two Schistosoma species have been shown to hybridize repeatedly, which triggered considerable parasi-tological and public health concern [69, 70]. Thus, future surveys are suggested to include molecular screening of schistosome infections [71]. Schistosoma bovis infec-tions of cattle have been confirmed from western Uganda [31]. Bulinus tropicus and B. forskalii are also intermedi-ate hosts for Schistosoma margrebowiei and Calicophoron microbothrium [72, 73], with B. forskalii transmitting a wide range of parasites in Ethiopia [74]. Several trematode infections have been reported in B. forskalii [75]. Loker et al. [76] detected cercariae of seven species from natu-rally infected snails in north-west Tanzania. Our study thus points to a significant concern since livelihoods of people in the crater lake region of western Uganda pre-dominantly depend on cows, sheep and goats, which are all susceptible to schistosomiasis and other trematodiases hosted by B. tropicus and B. forskalii [77]. The crater lakes are in close proximity to nature reserves and national parks that are home to one of the most diverse primate populations in Africa. It is thus noteworthy that zoonotic schistosomiasis is a significant concern at the human-wildlife interface that is currently largely underestimated [78], which makes the crater lake region further interest-ing for parasitological studies in addition to the relevance for increased intestinal schistosomiasis [79].
ConclusionsThis first detailed malacological study of the crater lakes systems in western Uganda revealed a dominance of Bulinus species that are either not known at all (B. tropi-cus and B. forskalii) or not known to act as intermediate hosts of S. haematobium, the causative agent of human urogenital schistosomiasis in this region of Africa (B. truncatus). The risk of contracting this form of schisto-somiasis is thus currently very low. However, potential sources for intermediate host species and known regions with high prevalence rates, have been identified in com-paratively close proximities to the study region (within a radius of c.250 km). The epidemiology of urogenital schistosomiasis is very dynamic and there is a potential for near-future occurrence in this part of Uganda. It is thus advisable to conduct more in-depth epidemiologi-cal studies in conjunction with the activities related to intestinal schistosomiasis. There is need for coordinated
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effort to document the genetic diversity of schistosome intermediate hosts from small-scale (in western Uganda) to large-scale (from Uganda as a country, to east Africa and the whole of Africa), so that an effort to eradicate the parasites via snail control from the natural system is based on informed grounds. A cautionary note is raised in terms of the veterinary importance of the gastropod species found. They both act as intermediate host for a variety of parasites including the species causing the majority of cases of livestock schistosomiasis, S. bovis. The impact of this finding is potentially of major impor-tance but currently unstudied in the region. Such studies are needed as well as investigations into factors driving the presence of hosts and parasites in regions and eco-systems so far largely neglected but with the potential of becoming major transmission sites. This is significant, especially under the projected climate changes that will shift altitudinal limits of one of the most notorious tropi-cal diseases that continues to be a major burden espe-cially in sub-Saharan Africa.
Supplementary informationSupplementary information accompanies this paper at https ://doi.org/10.1186/s1307 1‑019‑3811‑2.
Additional file 1: Figure S1. Photographs of Bulinus tropicus from Lake Mafuro (a, b) and a Bulinus species resembling B. globosus of Lake Kyaninga (c) showing variation in shell morphology. Both snails from Lake Mafuro are 11 mutation steps apart in the cox1 network.
Additional file 2: Figure S2. Bayesian inference phylogenetic tree for Bulinus spp. based on cox1. Specimens are given with locality information (country of origin and localities in some cases). The DNA preparation num‑bers are provided. Crater lake names are provided and the two specific clades of B. forskalii (Clade 1) and B. tropicus (Clade 2) are highlighted with light grey boxes. Crater lake populations are represented at the end of the branch by red boxes, while regional and non‑regional (= others) popula‑tions are demonstrated by green and grey boxes, respectively. Outgroup taxa are not shown. This tree is the full version of the collapsed tree in Fig. 3. Bayesian posterior probabilities (pp) are given for deeper nodes (when pp ≥ 0.5). The scale‑bar represents substitutions per site according to the applied model of sequence evolution. The number of individuals per haplotype is shown in parentheses for the two specific clades (for details see Figs. 4, 5).
Additional file 3: Table S1. Haplotype sequence matrix for Bulinus tropi-cus in 31 crater lakes of western Uganda. Abbreviations: NL, total number of haplotypes per lake; NH, total number of haplotype frequency (based on a 95% connection limit). For details of ‘lake codes’ see Table 1.
AbbreviationsAIDS: acquired immune deficiency syndrome; a.s.l.: above sea level; cox1: mito‑chondrial cytochrome c oxidase subunit 1 gene; CTAB: cetyl trimethyl ammo‑nium bromide; DNA: deoxyribonucleic acid; DRC: Democratic Republic of the Congo; MEGA: molecular evolutionary genetics analysis; NTDs: neglected tropical diseases; PAST: paleontological statistics; PCR: polymerase chain reac‑tion; UGSB: University of Giessen Systematics and Biodiversity collection.
AcknowledgementsWe thank the laboratory assistant Ms Silvia Nachtigall for her technical assistance in the laboratory. Support from colleagues at Mbarara University of Science and Technology (MUST) is appreciated, especially of Julius Bunny
Lejju for his assistance towards acquiring research permits and Bosco Kimenye for his company in the field as a driver. We are grateful for the Bulinus samples of Kahirimbi western Uganda from Joseph Jude Agaba. Paul‑Emile Desaulles, University of Lausanne, Switzerland is acknowledged for his help in the field for the material collected in 2016. The National Council for Science and Technology and Uganda Wildlife Authority kindly issued permits to conduct this study. Thies Geertz and Daniel Engelhard are gratefully acknowledged for their field work in 2010. The comments of three anonymous reviewers helped improving a previous version of the manuscript and are gratefully acknowledged.
Authors’ contributionsIT and CA conceived the study. IT did field work, produced and analyzed the molecular data and drafted the tables/figures. IT and CA drafted the initial manuscript, while the latter is the overall supervisor of the study. CC was involved in data analyses, drafting the manuscript and fine‑tuning the tables/figures. MCD and JT conducted significant parts of the field sampling. GKR was involved in planning and organizing the study and helped with permits and logistics in the field as well as in drafting the initial manuscript. All authors read and approved the final manuscript.
FundingIT is supported by a PhD scholarship of the German Academic Exchange Service (DAAD).
Availability of data and materialsData supporting the conclusions of this article are included within the article and its additional files. The newly generated sequences were submitted to the GenBank database under the accession numbers MN551500‑MN551585. The datasets generated and analysed during the present study are available in the University of Giessen Systematics and Biodiversity repository and are available upon reasonable request.
Ethics approval and consent to participateThis study was approved by Uganda National Council for Science and Technol‑ogy (UNCST) (research clearance reference number NS20ES).
Consent for publicationNot applicable.
Competing interestsThe authors declare that they have no competing interests.
Author details1 Department of Animal Ecology and Systematics, Justus Liebig University Giessen, Giessen, Germany. 2 Rwanda Wildlife Conservation Association, Kigali, Rwanda. 3 Department of Biology, Mbarara University of Science and Technol‑ogy, Mbarara, Uganda. 4 Department of Biology, Royal Museum for Central Africa, Leuvensesteenweg 13, 3080 Tervuren, Belgium. 5 Limnology Research Unit, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium.
Received: 29 April 2019 Accepted: 15 November 2019
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